The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the L XXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR F XXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound F XXLF and L XXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator L XXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in L XXLL motif binding parallels expansion and functional dominance of the NH 2-terminal transactivation domain in the steroid receptor subfamily.